Protein-protein interactions play a pivotal role in virtually every biological process, including the replication of pathogenic viruses in host cells. Due in part to their importance for virus replication, several interactions between viral proteins have been proposed as attractive targets for antiviral drug discovery, as the exquisite specificity of such interactions affords the possibility of interfering with them in a highly selective and effective manner (Loregian et al., 2002). We have been investigating one such interaction—that between the two subunits of the DNA polymerase of human cytomegalovirus (“HCMV”)—as a new drug target (Loregian et al., 2004a; Loregian et al., 2004b; Loregian et al., 2003). HCMV is a member of the DNA herpes virus family. HCMV has been isolated from saliva, urine, breast milk, blood, semen, and vaginal secretions. It can be transmitted in utero, despite the presence of high maternal antibody titers. Once infected, the individual conserves the virus in a latent or persistent form throughout life.
HCMV is an ubiquitous herpesvirus. Although it rarely causes symptomatic disease in immunocompetent individuals, it is responsible for a variety of severe diseases, including pneumonia, gastrointestinal disease and retinitis, in transplant recipients and in AIDS patients (Pass, 2001). HCMV is also a major cause of congenital malformation in newborn children, often resulting in deafness and mental retardation (Trincado et al., 2000). Antiviral agents currently licensed for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir, all of which inhibit the viral DNA polymerase). Ganciclovir and cidofovir are nucleoside analogs which function as DNA chain terminators, whereas foscarnet inhibits HCMV DNA polymerase through binding to its pyrophosphate binding site. However, these drugs are limited by their toxicities, pharmacokinetic drawbacks, and/viral resistance issues. Thus, there is considerable need for new anti-HCMV compounds.
Serological surveys indicate that most adults have been infected with HCMV. Following primary infection, which is almost always asymptomatic in people with normal immunity, the virus establishes latency. The virus is probably maintained at least in part in this latent state by immune surveillance mechanisms since immunosuppression frequently leads to reactivation of the virus. Reactivation of HCMV in immunosuppressed individuals can give rise to life-threatening disease.
HCMV infections are manifested in a variety of disease states. Infection with HCMV during pregnancy can lead to congenital malformation resulting in mental retardation and deafness. Infections of premature newborns can also result in significant morbidity. Although most infections of older immunocompetent individuals do not result in obvious disease, in young children they can sometimes be expressed as severe respiratory disease, and in older children and adults, they are sometimes expressed as anicteric hepatitis and mononucleosis.
HCMV pneumonitis is the most common single cause of death following bone marrow transplantation, and disseminated HCMV infection is a major cause of mortality and morbidity in patients with solid organ transplants or with AIDS. There is also evidence of a link between HCMV and atherosclerosis.
Like other herpes viruses, HCMV has a propensity to reactivate, particularly in immunosuppressed patients. Thus, HCMV infections present a major clinical problem for AIDS patients and other immunocompromised individuals such as organ transplant recipients and other patients receiving immunosuppressive drugs. Among AIDS patients, HCMV is the causative agent of certain invasive diseases such as retinitis, which is sight threatening, peripheral retinitis (an earlier form of the infection), esophagitis, and colitis.
In modern medical practice, HCMV is a significant pathogen whose ultimate control by means of immunization or drug therapy has become an important objective. So far, preliminary vaccination efforts have been unsuccessful, and no ideal therapeutic agent has been developed which can efficiently contain HCMV infection. Prophylaxis and therapy using HCMV immune globulins have met with only moderate success. In addition, therapeutic agents developed for treating HCMV infections have the common disadvantages of some type of toxicity to the host and the inability to rid the host of latent infection.
A major obstacle in developing suitable drugs possessing antiviral activity against HCMV is the ability to distinguish between the virus and the patient's own cells. HCMV, like other viruses, can only replicate by physically invading a cell and using the cell's biochemical pathways to make new viral proteins.
Because virus replication cycles are intimately connected with the functions of the host cell, there are few features peculiar to the virus that are not also present in the host. This makes selective attack on the virus difficult. Therefore, antiviral compounds generally represent a compromise between suppression of virus replication while minimizing adverse effects on the host.
Another obstacle to the inhibition of protein-protein interactions is that these interactions often involve a large surface area and multiple contacts (Archakov et al., 2003; Tsai et al., 1997). However, several studies have shown that relatively few residues within these large surfaces can drive binding (Cunningham and Wells, 1997) and that single substitutions in one subunit of a protein-protein interface can completely disrupt subunit interactions or nearly so (Eubanks et al., 2000; Imai et al., 2000; Koltzscher and Gerke, 2000; Lomax et al., 1998; Ramadevi et al., 1998; Sengchanthalangsy et al., 1999; Stenberg et al., 2000; Thomas et al., 1998). We have previously investigated this issue with the HCMV DNA polymerase, which consists of a 1242-residue catalytic subunit, UL54 (Cihlar et al., 1997; Kouzarides et al., 1987; Nishiyama et al., 1983), and a 433-residue accessory protein, UL44, which has been proposed to act as a processivity factor (Ertl and Powell, 1992; Weiland et al., 1994). We found that single substitutions in either UL54 or UL44 can drastically and specifically disrupt the interaction of these two proteins and their ability to synthesize long chains of DNA (Loregian et al., 2004a; Loregian et al., 2004b). Both UL54 and UL44 are essential for HCMV DNA replication (Pari and Anders, 1993; Pari et al., 1993; Ripalti et al., 1995) and the UL54-UL44 interaction is specific (Loregian et al., 2003). Additionally, a peptide corresponding to the C-terminal 22 residues of UL54 can both disrupt the physical interaction between UL54 and UL44 and specifically inhibit the stimulation of UL54 activity by the accessory protein (Loregian et al., 2003). Taken together these observations suggest that a small molecule could specifically inhibit the UL54-UL44 interaction and HCMV replication.
Although there are only a few examples of small molecules that disrupt protein-protein interactions and exert effects inside cells (reviewed in (Arkin and Wells, 2004; Cochran, 2000; Toogood, 2002)), we were encouraged by the recent identification of a small molecule (BP5) that specifically inhibits the physical interaction between the two subunits of herpes simplex virus type 1 (HSV-1) DNA polymerase, UL30 and UL42, which are homologous to UL54 and UL44, respectively, as well as HSV-1 replication in infected cell cultures (Pilger et al., 2004). Thus, the goal of this study was to determine if we could identify, via high throughput screening, compounds that could specifically inhibit the UL54-UL44 interaction. These studies led to the discovery of several small molecules with selective anti-HCMV activity.